PROJECT SUMMARY/ABSTRACT This application directly responds to the objective of RFA-AG-17-051. We have recently reported a positive correlation of the levels of pathogenic phosphorylated microtubule-associated protein tau (p-tau) in plasma- derived neuronal exosomes in Alzheimer's disease (AD) patients, suggesting their potential application for biomarkers and their implication as a novel machinery of spreading pathogenic molecules in the brain. However, the exact mechanisms regulating exosome biogenesis and secretion, and their contribution to the propagation of pathogenic molecules are poorly understood. Here we propose comprehensive approaches to delineate the molecular mechanisms that regulate biogenesis, secretion and trafficking of exosomes in different brain cell types (neurons, astrocytes and microglia) in vitro and in vivo. This project will (1) analyze the roles of the ESCRT (endosomal sorting complexes required for transport)-dependent pathway, combined with the ESCRT-independent (lipid- and tetraspanin-dependent pathways), for exosome biogenesis and secretion in murine and human neuronal cell types, (2) define the molecular machinery components responsible for exosome biogenesis and secretion in human induced pluripotent stem cells (hiPSCs) derived from AD patients, (3) characterize the exosome protein interactions with target cells for propagation using AD hiPSCs, and (4) characterize the trafficking of exosomes originated from specific neuronal and glial cell types in the central nervous system to periphery. We have assembled a group of collaborative investigators with established programs in cell biology, exosome biology, proteomics and animal models of neurodegenerative disorders. The proposed research work will develop a new understanding of exosomal biology and detailed functions of exosomes in progression of AD pathology. The findings gained from this research project will have a potential to discover new molecular targets for suppression of the disease spread via exosomes, and also address the National Alzheimer's Project Act plan to accelerate basic research toward development of AD therapeutics.